RNA interference (RNAi) has emerged as a powerful tool for basic biological studies as well as potential therapeutic agents. Numerous studies have demonstrated anti-HIV effects of short-interfering RNAs (siRNAs) or short-hairpin RNAs (shRNAs) targeting various HIV genes. However, constitutive gene silencing by administration of siRNAs or shRNAs may lead to various problems including off-target effects, emergence of escape virus mutants, and immunological complications. Therefore, conditional RNAi strategies that respond to the HIV derived or related factors would have profound implications in HIV research and therapy. At present, conditional expression of shRNAs from engineered promoters incorporating the trans-activating region (TAR) of HIV is the only major strategy that affords HIV-inducible RNAi. We propose to develop an alternative strategy for conditional RNAi responsive to the HIV proteins Tat or Rev based on our engineered RNA architecture that enable posttranscriptional activation of RNAi via allosteric hammerhead ribozyme. Specific Aims of the proposed research are as follows: Specific Aim 1: To engineer allosteric hammerhead ribozymes (aptazymes) that are activated by the HIV proteins Tat and Rev by incorporating natural and synthetic RNA ligands in the hammerhead ribozyme. Specific Aim 2: To demonstrate conditional gene silencing with engineered shRNAs incorporating the aptazymes in cultured cells. This R21 proposal will focus on demonstrating the feasibility of HIV Tat or Rev inducible gene silencing in human cell culture based on our original RNA architecture. If the project is successful, we hope to leverage the generated data to propose to further optimize the system to show inhibitory effects on HIV replication in cell culture and animal models. PUBLIC HEALTH RELEVANCE: RNA interference (RNAi) is an emerging technology with promising applications in gene therapy of various diseases including AIDS. The main goal of the proposed research is to develop the next generation RNAi platform that improves the efficacy of potential HIV gene therapy strategies by activating RNAi only after sensing viral proteins in the infected cells.